Damage to the limbal epithelial cells can be caused by chemical or thermal burns, the Stevens-Johnson syndrome, ocul cicatrIcial pemphigoid, contact lenses, severe infection, congenital aniridia, pterygium, herpes simplex epithelial disease contact lens induced keratopathy, neuroparalytic keratitis, drug toxicity, or multiple surgical procedures in the corneolim region and may lead to loss of limbal stem cells. The resulting so-called "limbal stem cell deficiency" is manifested by vascularization and chronic inflammation of the cornea, ingrowth of fibrous tissue, ulceration and corneal opacification. Transplantation of limbal tissue, supposedly including stem cells, from the limbus of the contralateral eye (if normal), or from a donor, can restore useful vision by providing stem cells which re-populate the cornea with normal epithelial cells. However, these procedures require large limbal grafts from the patient's healthy fellow eye and are not possible (except from a donor) in patients who have bilateral limbal stem cell deficiency. Using donor stem cells may result in rejection and requires prophylactic immunosuppression. Bioengineered corneal limbal stem cells and autograft tissue derived from the patient could circumvent these problems, but specific cell surface markers for limbal stem cells have not yet been developed. We will be guided by recently published methods, by finding for skin epidermal stem cells and by our preliminary studies. Three specific aims include: 1) identifying cell surface markers for corneal limbal stem cells. 2) establishing primary limbal stem cell cultures and evaluating the markers on cloned limbal stem cells to determine whether they correlate with in vivo stem cell surface markers. 3) isolating identified and marked corneal limbal stem cells using state of the art techniques for non-contact laser micromanipulation of cells, thereby providing a source of limbal stem cells for culture and further study. This work will advance our understanding of corneal limbal stem cell biology and may lead to the development of an RO1 proposal based on our findings. Such a proposal will include studies of the function of limbal stem cells in corneal epithelial healing; in epithelial migration and anchoring to underlying stroma; in differentiation and maturation of stem cells to transient amplifying cells, basal cells, and then mature surface cells; in interacting with recently described conjunctival epithelial stem cells. All such studies have previously been limited by lack of cell surface markers to localize stem cell population.